PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast–iPS–motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.     

Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis – vulnerability of lower motor neurons to proximal excitotoxicity

There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of individuals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.KEY WORDS: Motor neuron disease, Amyotrophic lateral sclerosis, Excitotoxicity, Lower motor neuron, Excitotoxin exposure     

CYP2E1 and Parkinson’s disease in a MPTP-induced C57BL/6 mouse model

Background

A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca²⁺ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.

Results

To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.

Conclusion

Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.     

Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons

Background

Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS.

Methodology/Principal Findings

We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model.

Conclusions/Significance

These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases.     

Differential effects of mutant SOD1 on protein structure of skeletal muscle and spinal cord of familial amyotrophic lateral sclerosis: Role of chaperone network

Protein misfolding is considered to be a potential contributing factor for motor neuron and muscle loss in diseases like Amyotrophic lateral sclerosis (ALS). Several independent studies have demonstrated using over-expressed mutated Cu/Zn-superoxide dismutase (mSOD1) transgenic mouse models which mimic familial ALS (f-ALS), that both muscle and motor neurons undergo degeneration during disease progression. However, it is unknown whether protein conformation of skeletal muscle and spinal cord is equally or differentially affected by mSOD1-induced toxicity. It is also unclear whether heat shock proteins (Hsp′s) differentially modulate skeletal muscle and spinal cord protein structure during ALS disease progression. We report three intriguing observations utilizing the f-ALS mouse model and cell-free in vitro system; (i) muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low level of soluble and absence of insoluble G93A protein aggregate, unlike in spinal cord, (ii) Hsp′s levels are lower in muscle compared to spinal cord at any stage of the disease, and (iii) G93ASOD1 enzyme-induced toxicity selectively affects muscle protein conformation over spinal cord proteins. Together, these findings strongly suggest that differential chaperone levels between skeletal muscle and spinal cord may be a critical determinant for G93A-induced protein misfolding in ALS.     

Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.     

Spinal Muscular Atrophy with Pontocerebellar Hypoplasia Is Caused by a Mutation in the VRK1 Gene

The spinal muscular atrophies (SMAs) are a genetically and clinically heterogeneous group of disorders characterized by degeneration and loss of anterior horn cells in the spinal cord, leading to muscle weakness and atrophy. Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH, also known as pontocerebellar hypoplasia type 1 [PCH1]) is one of the rare infantile SMA variants that include additional clinical manifestations, and its genetic basis is unknown. We used a homozygosity mapping and positional cloning approach in a consanguineous family of Ashkenazi Jewish origin and identified a nonsense mutation in the vaccinia-related kinase 1 gene (VRK1) as a cause of SMA-PCH. VRK1, one of three members of the mammalian VRK family, is a serine/threonine kinase that phosphorylates p53 and CREB and is essential for nuclear envelope formation. Its identification as a gene involved in SMA-PCH implies new roles for the VRK proteins in neuronal development and maintenance and suggests the VRK genes as candidates for related phenotypes.     

The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.     

Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1^G93A transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1^G93A astrocytes. Treatment of SOD1^G93A mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1⁺ oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Early Gene Expression Changes in Skeletal Muscle from SOD1G93A Amyotrophic Lateral Sclerosis Animal Model

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons. Familial ALS is strongly associated to dominant mutations in the gene for Cu/Zn superoxide dismutase (SOD1). Recent evidences point to skeletal muscle as a primary target in the ALS mouse model. Wnt/PI3 K signaling pathways and epithelial–mesenchymal transition (EMT) have important roles in maintenance and repair of skeletal muscle. Wnt/PI3 K pathways and EMT gene expression profile were investigated in gastrocnemius muscle from SOD1^G93A mouse model and age-paired wild-type control in the presymptomatic ages of 40 and 80 days aiming the early neuromuscular abnormalities that precede motor neuron death in ALS. A customized cDNA microarray platform containing 326 genes of Wnt/PI3 K and EMT was used and results revealed eight up-regulated (Loxl2, Pik4ca, Fzd9, Cul1, Ctnnd1, Snf1lk, Prkx, Dner) and nine down-regulated (Pik3c2a, Ripk4, Id2, C1qdc1, Eif2ak2, Rac3, Cds1, Inppl1, Tbl1x) genes at 40 days, and also one up-regulated (Pik3ca) and five down-regulated (Cd44, Eef2 k, Fzd2, Crebbp, Piki3r1) genes at 80 days. Also, protein–protein interaction networks grown from the differentially expressed genes of 40 and 80 days old mice have identified Grb2 and Src genes in both presymptomatic ages, thus playing a potential central role in the disease mechanisms. mRNA and protein levels for Grb2 and Src were found to be increased in 80 days old ALS mice. Gene expression changes in the skeletal muscle of transgenic ALS mice at presymptomatic periods of disease gave further evidence of early neuromuscular abnormalities that precede motor neuron death. The results were discussed in terms of initial triggering for neuronal degeneration and muscle adaptation to keep function before the onset of symptoms.     

Spinal Muscular Atrophy and the Antiapoptotic Role of Survival of Motor Neuron (SMN) Protein

Spinal muscular atrophy (SMA) is a devastating and often fatal neurodegenerative disease that affects spinal motor neurons and leads to progressive muscle wasting and paralysis. The survival of motor neuron (SMN) gene is mutated or deleted in most forms of SMA, which results in a critical reduction in SMN protein. Motor neurons appear particularly vulnerable to reduced SMN protein levels. Therefore, understanding the functional role of SMN in protecting motor neurons from degeneration is an essential prerequisite for the design of effective therapies for SMA. To this end, there is increasing evidence indicating a key regulatory antiapoptotic role for the SMN protein that is important in motor neuron survival. The aim of this review is to highlight key findings that support an antiapoptotic role for SMN in modulating cell survival and raise possibilities for new therapeutic approaches.     

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1^G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1^G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.     

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis-like Phenotypes in Mice

ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly, the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions (UBIs) in the cytosol. However, the role of the mismetabolism of TDP-43 in the pathogenesis of ALS with TDP-43 proteinopathies is unclear. Using the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbp(lx/-)) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle weakness/atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre-Tardbp(lx/-) mice with the ALS phenotypes. This study not only establishes an important role of TDP-43 in the long term survival and functioning of the mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.     

Molecular Chaperone Mediated Late-Stage Neuroprotection in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1^G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1^G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1^G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.     

Genetic Overlap between Apparently Sporadic Motor Neuron Diseases

Progressive muscular atrophy (PMA) and amyotrophic lateral sclerosis (ALS) are devastating motor neuron diseases (MNDs), which result in muscle weakness and/or spasticity. We compared mutation frequencies in genes known to be associated with MNDs between patients with apparently sporadic PMA and ALS. A total of 261 patients with adult-onset sporadic PMA, patients with sporadic ALS, and control subjects of Dutch descent were obtained at national referral centers for neuromuscular diseases in The Netherlands. Sanger sequencing was used to screen these subjects for mutations in the coding regions of superoxide dismutase-1 (SOD1), angiogenin (ANG), fused in sarcoma/translated in liposarcoma (FUS/TLS), TAR DNA-binding protein 43 (TARDBP), and multivesicular body protein 2B (CHMP2B). In our cohort of PMA patients we identified two SOD1 mutations (p.D90A, p.I113T), one ANG mutation (p.K17I), one FUS/TLS mutation (p.R521H), one TARDBP mutation (p.N352S), and one novel CHMP2B mutation (p.R69Q). The mutation frequency of these genes was similar in sporadic PMA (2.7%) and ALS (2.0%) patients, and therefore, our findings demonstrate a genetic overlap between apparently sporadic PMA and ALS.